CS246541B1 - Separating method of antracyclines - Google Patents

Description

The invention relates to a process for the isolation of anthracyclines, important anti-tumor antibiotics.

Said anthracyclines may be prepared either synthetically or by treatment with various species of actinomycetes, such as Streptomyces coeruleorubidus, Streptomyces peucetius, Actinomadara carminata.

The isolation procedures published so far (Process Biochemistry Vol. 14, No. 12, 1979; Folia Microbiol. 22, 275-285, 1977 et al.) Utilize conventional methods for recovering anthracyclines from fermentation broths by organic solvent extraction, isolation on ion exchangers, or a combination of both, precipitation methods based on the insolubility of the active substance in the respective solvent and subsequent crystallization and the like.

A disadvantage of these anthracycline isolation methods is the considerable amount of solvents, since multiple extractions of the entire volume of native soil are required, with the waste material usually containing a certain percentage of biologically active substances. Due to these two factors, the efficiency of the insulation process decreases. Not to be overlooked are the difficulties of manufacturing in handling large volumes of reaction mass.

The aforementioned disadvantages of anthracyclines with a 7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-naphthacenchinone skeleton, in particular daunomycin or adriamycin, their aglycones or other derivatives prepared by synthesis or fermentation, are eliminated by the process of isolation of anthracyclines according to the invention.

The principle of isolation of anthracyclines according to the invention consists in adding to the active substance solution an equivalent amount of a divalent transition metal salt sufficiently soluble in the medium, then adjusting the pH of the solution to 4 to 9, after removing the mother liquid medium the complex is dissolved and / or suspended in the solvent used for the next synthesis or isolation step, the resulting solution is optionally acidified with oxalic acid to a pH of 1 to 4 with stirring, whereupon the precipitated oxalate of the respective metal is separated and way. In the case of the isolation of daunomycin, the first extraction is carried out with butyl acetate.

If a raw solution of the active substance after fermentation is available, the essence of the process according to the invention consists in adding to this raw solution an equivalent amount of a divalent transition metal salt sufficiently soluble in the medium, then adjusting the pH of the solution to 4 to 9 After removing the parent liquid medium, the formed sparingly soluble metal complex together with the mycelium is suspended in the solvent used for the next isolation step.

The resulting suspension is acidified to pH 1-4 with stirring, the mycelium is separated and the pH is adjusted to pH 4-9 in the filtrate, the resulting sparingly soluble metal complex is separated and / or suspended in the solvent used for the next isolation step, the solution obtained is optionally acidified with oxalic acid to a pH of 1 to 4, with stirring, after which the oxalate of the corresponding metal is separated off and the filtrate of the active substance obtained is treated in a manner known per se. In the case of the isolation of daunomycin, the first extraction is carried out with butyl acetate.

A further advantage of the isolation of the anthracyclines according to the invention is that an equivalent of copper ions, preferably in the form of copper chloride or copper sulphate, is added to the crude active compound solution, thereby forming a metal complex with a stoichiometry of metal / drug = -1: 1 to 2.

The process for isolating anthracyclines of the invention is further characterized by adjusting the pH of the solution to 6.5 after addition of an appropriate amount of the salt of the metal concerned.

A further advantage of isolating anthracyclines after fermentation by the process according to the invention is characterized in that the resulting suspension of mycelium and the metal sparingly soluble complex is acidified with hydrochloric acid to a value of 1.3 to 1.5, thereby dissolving the metal complex and thereby allowing the active compound to be filtered off. no longer needed mycelium.

In the case of isolation of anthracyclines after fermentation, the process according to the invention is characterized in that after removal of the parent liquid medium including mycelium, the resulting metal complex solution or suspension is acidified with stirring with oxalic acid to pH 1.5 to dissolve the metal complex and precipitate the oxalate. metal.

Thus, the process of the invention utilizes the properties of 7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-naphthacenchinone derivatives to form sparingly soluble metal complexes with some transition metals (Biochim. Biophys. Acta 103, 25, 1975; Inorg. Biochem., Vol. 16, 91-107 (1982)) by binding the metal to the deprotonated form of anthraclinone, as shown in the following scheme: (an example of the binding of copper to daunomycin)

243541

.CCH,

0MQ / ^Cu

The process for the isolation of anthracyclines according to the invention consists in the possibility of quantitative precipitation of anthracycline-type substances obtained synthetically and fermentatively from fermentation broths, aqueous solutions and organic solvents in the form of a very sparingly soluble metal complex, in its separation and further processing in an already of the order of magnitude less than the volume from which the active substance was precipitated. This is particularly important from a production point of view. The solubility of a substance in a certain solvent under given conditions is, as a natural constant, an invariable quantity, and therefore the efficiency of its isolation must increase by quantitatively converting the substance to a much smaller volume. The method according to the invention utilizes this principle.

The total insolubility of the metal anthracycline complexes at pH 4-9 in fermentation broths in water or organic solvents allows reduction of the treatment volume at any stage of the isolation process. This creates much better conditions for the application of the classical extraction method, which can be part of the isolation process. In the case of sufficient purity of the metal-precipitated anthracyclines, either due to the sufficient selectivity of the production strain or previous operations, the extraction can be eliminated and after removal of the metal the active substance can be crystallized directly from the solution. The metal used to precipitate the complex (e.g., iron, zinc, copper, cobalt, nickel, etc.) can be easily removed by precipitating it in the form of a sparingly soluble salt (eg, oxalate, sulfide) and filtering it, or solvent during extraction (if this is done), where the precipitate of the complex remains in the aqueous phase, but the latter is less suitable for the frequent formation of stiff emulsions.

The method of isolation of anthracyclines according to the invention follows from the following block diagram:

The block diagram of the quantitative precipitation of anthracyclines (type 7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-naphthacenchinone) needs to be appropriately modified to isolate the individual derivatives; for example, in the isolation of daunomycin from a fermentation broth, removal of the metal in the first phase of the isolation is not expedient.

In addition to the advantages already mentioned, the precipitation of anthracyclines in the form of the metal sparingly soluble complexes according to the invention also has the advantage that, in the isolation of the active substances from native soils, the nutrient solution can be removed at the beginning of the isolation. Moreover, in all cases of isolation of the anthracyclines according to the invention from native soil by this method, a considerable acceleration of the mycelium separation by filtration on kieselguhr filters has been found, which then proceeds without renewing the kieselguhr surface. The separation of the metal complex from a solution which is no longer mycelium facilitates the newly discovered and previously unpublished property of the metal complex, namely its ability to sediment to a third volume within 6 to 7 hours. The liquid layer above the metal complex sediment can be decanted and any subsequent centrifugation or filtration of the metal complex can be performed only in one third of the original volume from which the metal complex was precipitated. This greatly simplifies production equipment and reduces production costs.

Calculation of the isolation examples of compounds according to the invention containing a 7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-naphthacenedione skeleton in their molecule can be very comprehensive, since the method of quantitative precipitation of metal anthracycline complexes can be used in all insulation methods in which it is expedient to quantitatively convert the anthracycline into a small volume, for example in isolating anthracyclines on an ion exchanger. The process according to the invention makes it possible to reduce the volume of the liquid from which the active substance is adsorbed on the ion exchanger; it makes it possible to quantitatively obtain anthracycline from a chelate and then isolate it from a minimum volume. The process according to the invention can be applied in extraction extraction processes. The combination of the "classical" anthracycline isolation methods with the isolation method of the invention can be a wide variety.

The following examples illustrate the method of isolation of the anthracyclines of the present invention, but do not limit it in any way.

He did

Isolation of daunomycin

A slight excess of the metal salt (copper (II) chloride, zinc sulphate) is dissolved in 1000 ml of fermentation broth immediately after the fermentation so that the solution contains a metal equivalent to form a complex with stoichiometry of metaunomycin = 1: 1 to 2. pH) and the pH is adjusted to 6.5 by addition of concentrated ammonia. After brief mixing, the empty soil is filtered off on kieselguhr filter, the mycelium with the precipitated metal complex is washed on the filter with distilled water until the filtrate flows colorless. This removes the excess metal used to precipitate the complex (at pH values above 6.0, 7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-naphthacenchinone derivatives form metal complexes with metal / anthracycline stoichiometry = 1 : 2). The precipitated complex mycelium is transferred to a beaker where it is stirred in an amount of distilled water to form a stirrable suspension and the pH is adjusted to 1.3-1.4 by the addition of concentrated hydrochloric acid (HCl). The suspended mycelium was stirred at room temperature for 30 minutes. After this operation, the extracted mycelium is filtered on a diatomaceous earth filter and washed on the filter with acidified distilled water (HCl) until the filtrate flows colorless. To displace the last active substance residue from the filter cake (mycelium), it is possible at the end of the filtration to wash the mycelium with a small amount of ethanol into the filtrate. In the filtrate, the pH is adjusted to 6.5 by the addition of concentrated ammonia and the re-precipitated metal complex of daunomycin is appropriately separated - after sedimentation of the complex to one third volume and decantation - by centrifugation, or better filtration on kieselguhr filter. filter. All the anthracycline-like material contained in the original fermented soil, in the form of an insoluble metal complex, was obtained quantitatively by the above operations, the wet precipitate volume being 5 to 8% by volume of the original soil volume. Further operations with this preparation (dissolution, extraction, crystallization, etc.) are then carried out in the minimum volumes given by the need to handle them. The preparation obtained contains 98 to 99% of daunomycin from the original amount determined in native soil.

Suspend the wet or dry complex in a minimum amount of distilled water and add a slight excess of equivalent oxalic acid to the metal while stirring and cool the solution to 10 to 15 ° C. After stirring for 10 minutes, the precipitated copper oxalate is filtered off and the filtrate is extracted twice with an equal volume of butyl acetate in a separatory funnel or extraction column. Daunomycinone is transferred to butyl acetate, the aqueous phase containing daunomycin is extracted twice with an equal amount of n-butanol. In the Butanol extract, after combining the two portions, adjust the pH to 6.0 to 7.0 and filter it on a diatomaceous earth filter. The aqueous phase is additionally precipitated in the filtrate, which is separated and then the butanolic extract is evaporated to dryness under reduced pressure. The crude daunomycin obtained is then recrystallized in an appropriate manner (for example from a mixed solvent of methanol: ethanol: HCl: chloroform in a ratio of 3.5: 60: 70). Yield: 91% of daunomycin hydrochloride based on daunomycin content in native soil, mp 175-176 ° C .

Example 2

Isolation of daunomycin

In 1000 ml of fermentation broth, the pH is adjusted to 1.5 with concentrated hydrochloric acid after the fermentation is complete and stirred vigorously at 20 ° C for 30 minutes. Then the soil is filtered on kieselguhr and the mycelium on the filter is washed with acidified water (HCl, pH 2) until the filtrate flows colorless. Copper chloride is dissolved in the filtrate in an amount such that the solution contains a slight excess of copper equivalent to form a complex with a stoichiometry of Cu / daunomycin = 1: 1 and adjusts the pH to 6.5. The precipitated daunomycin Cu complex is further processed as in Example 1 and 2, respectively. 3.

Example 3

Daunomycin isolators

The isolation procedure is the same as in Example 1 until a damp metal daunomycin metal complex is obtained, the processing of which is as follows: the damp complex is drained on the diatomaceous earth by washing with n-butanol, then the complex is dissolved in n-butanol in which a slight excess of oxalic acid to bind the metal used to precipitate the complex. The precipitated oxalate is filtered off and the butanolic filtrate is treated in the same manner as the butanolic extract in Example 1 (pH adjustment, kieselguhr filtration, aqueous phase separation, evaporation, recrystallization).

Yield: 90-92% of daunomycin hydrochloride, m.p. 175-176 ° C.

Example 4

Daunomycinone insulators

A slight excess of copper equivalent (in the form of copper chloride) is dissolved in 1000 ml of fermentation broth to form a complex with copper / daunomycin stoichiometry = 1: 1 to 2 (the weight is determined by the number of units of active substance determined) and pH is adjusted by adding concentrated ammonia. to 6.5 and after brief mixing, the empty soil is filtered on kieselguhr; the precipitated complex mycelium is washed on the filter with distilled water. Thereafter, the filter cake is stirred in a minimum amount of distilled water and adjusted to pH 1.3 with concentrated hydrochloric acid with stirring. The suspension is heated to 85 ° C in a water bath; with stirring at this temperature, the acid hydrolysis (cleavage of daunosamine) takes place for 45 minutes. The suspension was then cooled to 5-10 ° C and allowed to stand at this temperature for 1 hour. After filtering off the mycelium with precipitated daunomycinone, the filter cake is extracted on the diatomaceous earth by stirring with 300 ml of acetone, the extracted mycelium is filtered off and washed with acetone into the filtrate so that the filtrate flows colorless. A slight excess of the equivalent amount of oxalic acid to dissolve the metal is dissolved in the filtrate and after stirring for 20 minutes the precipitated copper oxalate is filtered off. The filtrate containing daunomycinone is evaporated to dryness in a vacuum evaporator. Crude daunomycinone is obtained in a 95% yield (based on the determined amount of active ingredient units in the native soil), m.p. 215 ° C. Recrystallization of the obtained daunomycinone was carried out from glacial acetic acid.

Example 5

Isolation of adriamycin

According to Example 1, adriamycin was isolated from the fermentation broth. The isolation procedure is identical to that of obtaining the aqueous phase after extraction with butyl acetate. Extract the aqueous phase twice with an equal amount of chloroform. The chloroform extract is dried over sodium sulfate and then evaporated to dryness under reduced pressure. The crude adriamycin obtained is recrystallized from a solvent mixture of methanol: ethanol. HCl: chloroform. Yield: 90% adriamycin hydrochloride based on adriamycin content in the m.p. 205-208 ° C.

Example

Isolation of adriamycin

After the synthesis of adriamycin from daunomycin, adriamycin was isolated from the reaction mixture as an insoluble copper complex by adding an equivalent amount of copper sulfate and adjusting the pH to 6.5. The treatment of the complex is carried out according to points 1 and 5.

Example 7

Isolation of daunomycin

Following the procedure of Example 1, the copper complex of daunomycin copper sulfate was used with the same yield of daunomycin as in Example 1.

248 541

Example 8

Isolation of daunomycin

The procedure of Example 1 was used

Claims (7)

Method for the isolation of anthracyclines having a skeleton of 7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-naphthacenchinone, in particular daunomycin or adriamycin, their aglycons or other derivatives prepared by synthesis or fermentation, characterized in that an equivalent amount of the bivalent transition metal salt sufficiently soluble in the medium is added to the crude active compound solution after synthesis, then the pH of the solution is adjusted to 4-9, and after removal of the parent liquid medium the resulting sparingly soluble metal complex is dissolved and / or suspended in the same the solvent used in the synthesis or the solvent used for the next step of the synthesis, the resulting solution is optionally acidified with oxalic acid to a pH of 1 to 4 with stirring, whereupon the precipitated oxalate of the corresponding metal is separated and the obtained active substance filtrate is worked up in a known manner or of the mother liquor After fermentation, an equivalent amount of a divalent transition metal salt sufficiently soluble in the medium is added directly thereafter, the pH of the solution is adjusted to 4 to 9, after removal of the liquid mother medium, the formed sparingly soluble metal complex is suspended together with mycelium in water or solvent. used for the next isolation step, the resulting suspension is acidified to pH 1-4 with stirring, then the mycelium is separated and the pH is adjusted to pH 4-9 in the filtrate, the formed sparingly soluble metal complex is separated and / or suspended in the same solvent The solution obtained is optionally acidified with oxalic acid to a pH of 1 to 4 with stirring, after which the precipitated oxalate of the metal in question is separated and gives a metal complex of daunomycin zinc sulfate with the same yield of daunomycin as in Example 1. The active substance filtrate of the invention is worked up in a manner known per se, but in the case of isolation of the daunomycin, the first extraction is always carried out with butyl acetate. 2. A process according to claim 1, wherein an equivalent of copper (I) ions, preferably in the form of copper (II) chloride or sulfate, is added to the crude active compound solution to form a metal complex with a metal / drug stoichiometry = 1: 1 to 2. 3. The process of claim 1 wherein the pH of the solution is adjusted to a value of 6.5 after addition of an appropriate amount of salt of the metal concerned. 4. The process of claim 1 wherein the resulting suspension of the mycelium and the sparingly metal complex is acidified with hydrochloric acid to a pH of 1.3 to 1.5. 5. A process according to claim 1, characterized in that, after removal of the parent liquid medium, the resulting metal complex solution or suspension is acidified with stirring with oxalic acid to a pH of 1.5. 6. The process of claim 1, wherein after removal of the parent liquid medium including mycelium, the resulting metal complex solution or suspension is acidified with mineral acid to pH 1.5 with stirring, and the resulting solution is saturated with hydrogen sulfide. 7. The process of claim 1, wherein after removal of the parent liquid medium including mycelium, the resulting metal complex solution or suspension is acidified to 1.5 with stirring with hydrochloric acid, and the resulting solution is extracted with butyl acetate.